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. 2014 Apr;32(4):364-72.
doi: 10.1038/nbt.2858. Epub 2014 Mar 16.

Development and function of human innate immune cells in a humanized mouse model

Anthony Rongvaux  1 , Tim Willinger  1 , Jan Martinek  2 , Till Strowig  1 , Sofia V Gearty  3 , Lino L Teichmann  4 , Yasuyuki Saito  5 , Florentina Marches  6 , Stephanie Halene  7 , A Karolina Palucka  6 , Markus G Manz  5 , Richard A Flavell  8
Affiliations

Development and function of human innate immune cells in a humanized mouse model

Anthony Rongvaux et al. Nat Biotechnol. 2014 Apr.

Erratum in

Abstract

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

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Figures

Fig. 1
Fig. 1. Efficient engraftment of human hematopoietic cells in MI(S)TRG mice
X-ray pre-conditioned newborn mice of the indicated strains were engrafted by intra-hepatic injection of 100,000 human fetal liver CD34+ cells. Human engraftment (the percentage of hCD45+ cells among all (mouse and human) CD45+ cells) was measured in the blood 7-9 weeks later, and in the BM 10-12 weeks later. a, Representative flow cytometry analysis of the frequency of mouse and human CD45+ cells in the blood and BM of the indicated recipient mice. Numbers next to gated areas indicate percentages among total CD45+ cells. b, Blood engraftment from 19 independent experiments. In each experiment, a single fetal liver CD34+ cell sample was split and injected into mice of the respective strains. Each symbol represents an individual mouse and the red bars indicate mean values (n=56-155; ns, not significant; * p<0.05 Tukey test, see Supplementary Fig. 2a for a complete statistical analysis). The gray horizontal line indicates 10% hCD45+ cells. c, Engraftment in the BM of a representative subset of mice (Supplementary Fig. 2c) from panel b (n=12-16; * p<0.05 Tukey test; see also Supplementary Fig. 2d-e). d, Representative flow cytometry analysis of hCD45+ cell engraftment in the blood and BM 3 months after intra-hepatic injection of 200,000 fetal liver CD34+ cells into non-irradiated newborn MISTRG mice. e, Human CD45+ cell engraftment levels in the blood and BM of MISTRG mice transplanted as in d (n=16).
Fig. 2
Fig. 2. MI(S)TRG mice support efficient myeloid cell development in lymphoid and non-lymphoid tissues
a, Percentages of human myeloid cells (hCD33+) among human hematopoietic cells (hCD45+) in the blood of the indicated recipient mice, engrafted as newborns by intra-hepatic injection of fetal liver CD34+ cells after X-ray preconditioning. Each symbol represents an individual mouse and the red bars indicate mean values (n=20-113; statistical analysis is shown in Supplementary Fig. 3a). b, Composition of human white blood cells in the same mice as in a (n=20-113 mice/group; n=8 human donors; error bars indicate SEM). c, Immunohistological staining of human myeloid cells (hCD68+) in non-lymphoid tissues of the indicated recipient mice. The black bar represents 20 μm, and the images shown are representative of at least three mice analyzed per group. d-e, Representative flow cytometry analysis (d) and frequencies (e) of human monocyte subsets, identified by expression of CD14 and CD16 among hCD45+CD33+ cells in the blood of recipient mice (n=8-12 mice/group; error bars indicate SEM). Dot plots in d are gated on CD33hiSSClo cells to show the subset distribution among monocytic cells. f, Human monocytes in the blood of MISTRG recipients and human monocytes from a human donor were stained with the indicated antibodies. Staining with isotype control antibodies is shown in red and specific antibodies in blue.
Fig. 3
Fig. 3. Monocytes in MI(S)TRG mice are functional
a-b, Cytokine production by human monocytes isolated from the BM of MITRG recipients and stimulated overnight in vitro with 100 ng/ml LPS (a) or 10 μg/ml R848 (b) (error bars indicate SD of triplicates; representative of 3 independent experiments) was measured by ELISA. c, In vitro phagocytosis of GFP-expressing E.coli by human cell subsets, identified by flow cytometry gating, in the blood of MITRG mice (n=7). d-f, Mice were injected with LPS (d; 90 min, n=15-18), or infected with Listeria monocytogenes (e; day 2, n=6-15) or influenza A/PR8 H1N1 (f; day 3, n=3-5). Cytokine production was measured by ELISA (in the serum) or by RT-PCR (in the lung). (d) p-value calculated by unpaired Student's t-test on log10-transformed values; (e,f) p-values calculated by one-way ANOVA followed by Tukey posthoc test (* p<0.05).
Fig. 4
Fig. 4. Human NK cells develop efficiently in MISTRG mice
a, Quantitative RT-PCR analysis of human IL-15 and IL-15Rα mRNA expression in the liver of engrafted NSG, MITRG, and MISTRG mice (n=7-8; p-values calculated by one-way ANOVA; *, p<0.05 Tukey post hoc test). Expression was normalized to mouse Hprt. b, Quantitative RT-PCR analysis of human IL-15 and IL-15Rα mRNA expression in indicated human cell populations purified from bone marrow of engrafted MITRG mice (n=4-5, error bars indicate SEM). Expression was normalized to human HPRT and is shown relative to hCD14+hCD16- cells. c-d, Representative flow cytometry analysis (gated on hCD45+mCD45- cells, lymphocyte gate; numbers next to outlined areas indicate percentages of cells) (c) and absolute number or frequency (d) of human NK cells (hNKp46+ hCD3-) in engrafted NSG, MITRG, and MISTRG (n=8-16; p-values calculated by one-way ANOVA; *, p<0.05 Tukey post hoc test). e-f, Representative flow cytometry analysis (e) of human liver monocytes/macrophages (upper panel, gated on hCD33+ cells) and NK cells, and absolute numbers (f) of human liver NK (hNKp46+hCD3-) and T cells (hCD3+, shown as control) in engrafted MISTRG mice either left untreated or treated for 3 consecutive days with liposome-encapsulated clodronate to deplete phagocytic cells (n=8; p-value calculated by unpaired Student's t-test; ns, not significant). In a,d,f, each symbol represents an individual mouse. Black bars represent the mean. Results are combined from two (a, e f), three (b), or four (c, d) experiments.
Fig. 5
Fig. 5. Human NK cells in MITRSG mice are fully functional
a-b, Intracellular perforin expression by human liver NK (hNKp46+hCD3-) and T cells (hCD3+) from engrafted NSG and MISTRG mice (n=3; p-value calculated by unpaired Student's t-test). MFI, mean fluorescence intensity. c, Violet-labeled LCL721.221 (HLA class I negative) and LCL721.45 (HLA class I positive) cells were injected i.v. in a 1:1 ratio, and the proportions of HLA class I positive or HLA class I negative cells among Violet-labeled cells recovered 12 hours later in the spleen, were used to calculate specific NK cell-mediated lysis (n=8, p-value calculated by unpaired student's t-test). d, Quantitative RT-PCR analysis of human IFN-γ mRNA expression in the liver of NSG and MISTRG mice 2 days after Listeria infection (n=8-9, p-value calculated by unpaired student's t-test). Expression was normalized to mouse Hprt. e-f, Frequency (e) and representative flow cytometry analysis (f) of IFN-γ-expressing and degranulating (CD107a+) human liver NK cells from either uninfected or Listeria-infected NSG and MISTRG mice (n=4-11; p-value calculated by one-way ANOVA). In (b-e), each symbol represents an individual mouse. Black bars represent the mean. Results are representative of or combined from two experiments.
Fig. 6
Fig. 6. Infiltration and growth of a tumor in MISTRG mice
The human melanoma cell line Me290 was subcutaneously implanted in the flank of engrafted or non-engrafted NSG and MI(S)TRG mice. Where indicated mice were treated with the VEGF-inhibitor Avastin™ every two days, starting on the day of tumor injection. The tumors were measured and dissected for analysis 11 days later. a, Inflitration of human hematopoietic cells in the tumor, determined by the expression of PTPRC mRNA (encoding CD45) and ITGAM mRNA (encoding CD11b) (n=6-7; p-value calculated by unpaired Sudent's t-test). b and d, Representative immunohistochemistry images of human myeloid cell markers in tumors from NSG, MISTRG and human patients (the white scale bars indicate 100 μm). c, Quantification of the density of CD163+ cells in tumors (n=3 samples/group, 3 slides counted/sample). e-f, Representative pictures (e) and volume (f) of the tumors in the indicated groups of mice (n=7-24 mice/group). p-values were calculated by Student's t-test (a) or by one-way ANOVA (c, e) followed by Tukey posthoc test (* p<0.05).
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Comment in

  • New models of human immunity.
    Spits H. Spits H. Nat Biotechnol. 2014 Apr;32(4):335-6. doi: 10.1038/nbt.2871. Nat Biotechnol. 2014. PMID: 24714481 No abstract available.

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